CCR 7 Ligands Are Required for Development of Experimental Autoimmune Encephalomyelitis through Generating IL-23-Dependent Th17 Cells This information is current as of September 24, 2021. Taku Kuwabara, Fumio Ishikawa, Takuwa Yasuda, Kentaro Aritomi, Hideki Nakano, Yuriko Tanaka, Yayoi Okada, Martin Lipp and Terutaka Kakiuchi J Immunol 2009; 183:2513-2521; Prepublished online 22 July 2009; Downloaded from doi: 10.4049/jimmunol.0800729 http://www.jimmunol.org/content/183/4/2513 http://www.jimmunol.org/ References This article cites 40 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/183/4/2513.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CCR 7 Ligands Are Required for Development of Experimental Autoimmune Encephalomyelitis through Generating IL-23-Dependent Th17 Cells1

Taku Kuwabara,* Fumio Ishikawa,* Takuwa Yasuda,2* Kentaro Aritomi,*‡ Hideki Nakano,3* Yuriko Tanaka,* Yayoi Okada,* Martin Lipp,§ and Terutaka Kakiuchi4*†

CCL19 and CCL21 are thought to be critical for experimental autoimmune encephalomyelitis (EAE) induction, but their precise role is unknown. We examined the role of these in inducing EAE. C57BL/6 mice lacking expression of these chemokines (plt/plt mice) or their receptor CCR7 were resistant to EAE induced with myelin oligodendrocyte glyco- peptide 35–55 (MOG35–55) and pertussis toxin. However, passive transfer of pathogenic T cells from wild-type mice induced EAE in plt/plt mice, suggesting a defect independent of the role of CCR7 ligands in the migration of immune cells. Downloaded from

Examination of draining (DLN) cells from MOG35–55-immunized plt/plt mice found decreased IL-23 and IL-12 production by plt/plt dendritic cells (DCs) and a concomitant defect in Th17 cell and Th1 cell generation. In contrast, production of the Th17 lineage commitment factors IL-6 and TGF-␤ were unaffected by loss of CCR7 ligands. The adoptive transfer of in vitro-generated Th17 cells from DLN cells of MOG35–55-immunized plt/plt mice developed EAE in wild-type recipient mice, whereas that of Th1 cells did not. Pathogenic Th17 cell generation was restored in plt/plt DLNs with the addition of exogenous IL-23 or CCL19/CCL21 and could be reversed by inclusion of anti-IL-23 mAb in cultures. Exogenous http://www.jimmunol.org/ CCL19/CCL21 induced IL-23p19 expression and IL-23 production by plt/plt or wild-type DCs. Therefore, CCR7 ligands have a novel function in stimulating DCs to produce IL-23 and are important in the IL-23-dependent generation of pathogenic Th17 cells in EAE induction. The Journal of Immunology, 2009, 183: 2513–2521.

xperimental autoimmune encephalomyelitis (EAE)5 is an reactivation upon encountering endogenous Ag leading to nerve autoimmune disease of the CNS in mouse and rat that demyelination. Th17 cells, a helper CD4ϩ T cell lineage generated E serves as a disease model for human multiple sclerosis by IL-6, TGF-␤, and IL-23, are the pathogenic cells in EAE and (1). EAE is induced through sensitization with neuroantigens such are capable of inducing EAE in recipient mice by passive transfer by guest on September 24, 2021 as myelin oligodendrocyte glycoprotein (MOG) that activates neu- (2). The activation and activity of pathogenic T cells appear de- roantigen-reactive T cells in the peripheral lymphoid organs. Sub- pendent on the coordinated migration of several cell types, a phe- sequent migration of these T cells into the CNS leads to their nomena regulated by chemokines (3). Indeed, many chemokines have been shown to be critical for the development of EAE, mainly in the context of recruitment of immune cells into the CNS (4). *Department of Immunology and †Advanced Medical Research Center, Toho Uni- versity School of Medicine, Tokyo, Japan; ‡Department of Orthopedic Surgery, Jun- Chemokines may regulate induction of pathogenic T cells inde- tendo University School of Medicine, Tokyo, Japan; and §Department of Molecular pendent of their role in migration of immune cells but their role in Tumor Genetics and Immunogenetics, Max-Delbruck Center for Molecular Medicine, inducing pathogenic T cells in EAE is unclear. Berlin, Germany Using mutant mice lacking the expression of CCL19 and Received for publication March 6, 2008. Accepted for publication June 21, 2009. CCL21 ( plt/plt mice), we previously showed that these CC che- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance mokine ligands, which share the CCR7, not only play a critical role with 18 U.S.C. Section 1734 solely to indicate this fact. in the migration of naive T cells and dendritic cells (DCs) into 1 This work was supported in part by Project Research of Toho University School of secondary lymphoid organs, but also regulate in vivo T cell re- Medicine (to T.Ku., F.I., and Y.O.), the Research Promotion Grants from Toho Uni- sponses to an Ag (5–8). Based on these findings, we hypothesized versity Graduate School of Medicine (Grant 05-02 to T.Ka., Grant 07-02 to T.Ku., and Grant 08-02 to Y.T.), and Grants-in-Aid for Scientific Research from the Japan that these chemokines regulate not only the migration of patho- Society for the Promotion of Science to T.Ka. (Grants 17590900 and 19591013), to genic T cells into the CNS, which was suggested previously (9), T.Ku. (Grant 18790605), and to Y.K. (Grant 19790695). but also the induction of pathogenic T cells. In the present study, 2 Current address: Laboratory for Immunogenetics, RIKEN Research Center for Al- we examined the role of the CCL19 and CCL21 chemokines in the lergy and Immunology, Yokohama 230-0045, Japan. induction of EAE using plt/plt mice. Deficiency in the expression 3 Current address: Laboratory of Respiratory Biology, National Institute of Environ- mental Health Sciences. National Institutes of Health, 111 T.W. Alexander Drive, of these chemokines was protective against development of EAE Building 101, E244, Research Triangle Park, NC 27709. due to a defect in IL-23-dependent induction of Th17 cells; how- 4 Address correspondence and reprint requests to Dr. Terutaka Kakiuchi, Department ever, passively transferred pathogenic T cells could initiate EAE in of Immunology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143-8540, Japan. E-mail address: [email protected] plt/plt mice. Thus, chemokines CCL19 and CCL21 are crucial for the induction of pathogenic T cells, independent of their role in 5 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyeli- tis; DC, ; BMDC, bone marrow-derived DC; LN, lymph node; DLN, immune cell migration. In this study, we provide the first descrip- draining LN; MOG, myelin oligodendrocyte glycoprotein; rm, recombinant mouse; tion of a novel function of these CCR7 ligands, namely, the stim- WT, wild type; PT, pertussis toxin. ulation of DCs to produce IL-23, resulting in the generation of Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 Th17 cells. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0800729 2514 ROLE OF CCL19/21 IN IL-23 PRODUCTION AND EAE DEVELOPMENT

Materials and Methods real-time PCR was conducted using TaqMan Expression Assay Kits Mice (Applied Biosystems), Mm00434165_m1 for IL-12 and Mm00518984_m1 for IL-23 on an Applied Biosystems Prism 7000 Sequence Detector Sys- C57BL/6 mice were obtained from Charles River Laboratories Japan and tem. GAPDH was used as an endogenous reference for normalization. C57BL/6-plt/plt and C57BL/6-CCR7Ϫ/Ϫ mice were bred and kept at the Quantitative real-time PCR experiments were repeated twice in triplicate. Toho University School of Medicine animal facility (7) under specific To analyze the expression of IL-23p19 mRNA and IL-12p35 mRNA in ϩ pathogen-free conditions in accordance with the institutional guidelines. DLN DCs, CD11c cells were enriched from DLN cells using a magnetic All animal experiments were approved by the institutional review board. positive selection kit (BD Biosciences). Mice were used at 8–12 wk of age. Where indicated, CD45.1ϩ C57BL/ 6-Ly5.1 mice were also used. These mice were maintained in our animal Bone marrow-derived DCs (BMDCs) facility. The peripheral lymph node (LN) was much smaller in plt/plt mice than in wild-type (WT) mice (5). The mean cell number in an experiment BMDCs were prepared from bone marrow of naive C57BL/6 mice as pre- ϫ 6 was 4.2 ϫ 105 cells/LN in the inguinal and axillary LNs from six naive viously described (10). Where indicated, 4 10 cells in 10 ml of RPMI plt/plt mice, whereas 1.5 ϫ 106 cells/LN in naive WT mice. Nine days after 1640 containing rmGM-CSF (PeproTech/Tebu) were pulsed with rm- EAE induction as described below, the mean cell numbers were 6.7 ϫ 106 CCL19 or rmCCL21 (R&D Systems). and 2.1 ϫ 107 cells/LN in plt/plt and WT mice, respectively. The LN size was always macroscopically monitored before preparing LN cells. Results Reagents plt/plt mice lacking expression of CCL19 and CCL21 are resistant to EAE induction MOG35–55 peptide was prepared and provided by Dr. S. Imajoh-Ohmi (In- stitute of Medical Science, University of Tokyo, Tokyo, Japan). Allophy- To examine the role of the CCR7 ligands CCL19 and CCL21 in cocyanin- or FITC-labeled anti-CD4 (RM4-5, IgG2a, ␬), PE-labeled anti- EAE induction, C57BL/6 WT and C57BL/6-plt/plt mice were im- Downloaded from ␬ ␬ IL-17 (TC11–18H10, IgG1, ), PE-labeled rat IgG1 (R3-34, IgG1, )asan munized s.c. with MOG peptide in CFA on day 0 and injected isotype control, and anti-IFN-␥ mAb (XMG1.2, IgG1, ␬) were purchased 35–55 from BD Biosciences. Anti-IL-23 mAb (G23-8, IgG1, ␬), anti-IL-4 mAb i.v. with PT on days 0 and 2. As shown in Fig. 1A (left), WT mice (11B11, rat IgG1, ␬), anti-CD45.1 mAb (A20, mouse IgG2a, ␬), and anti- developed EAE with 100% disease incidence with onset at day 14, CD45.2 mAb (104, mouse IgG2a, ␬) were from eBioscience. Recombinant whereas plt/plt mice failed to develop EAE during the 42 days mouse (rm) GM-CSF was purchased from PeproTech/Tebu. rIL-12, rIL- following immunization. Confirming the requirement for CCR7 23, rmCCL19, and rmCCL21 were from R&D Systems. rmIL-6 and ligand in EAE development, similarly treated CCR7Ϫ/Ϫ mice did TGF-␤ were obtained from eBioscience. http://www.jimmunol.org/ not develop EAE (Fig. 1A, right). Histological analysis of the lum- EAE induction bar spinal cord from WT mice 21 days after immunization clearly ␮ demonstrated cellular infiltration in the spinal parenchyma, as Mice were immunized s.c. on the flanks on day 0 with 150 gofMOG35–55 peptide in CFA containing 5 mg/ml H37RA (Difco Laboratories). Two shown in Fig. 1B. No such infiltration was observed in the spinal hundred nanograms of pertussis toxin (PT; List Laboratories) was injected cord from plt/plt mice, consistent with the lack of EAE develop- i.v. on days 0 and 2. Mice were monitored for EAE development and ment (Fig. 1B). graded on a clinical scale of 0–5: 0, no disease; 1, limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4, hind and fore limb paralysis; and 5, That EAE did not develop in plt/plt mice might be due to the severe morbidity. For passive transfer of EAE, donor mice were immu- failure of pathogenic T cells to migrate into the CNS because of

nized as described above. Nine days later, draining LN (DLN) cells were the lack of CCR7 ligand expression, as suggested previously (9). by guest on September 24, 2021 ϫ 6 ␮ cultured at 4 10 cells/ml with 10 M MOG35–55 peptide for 3 days in To examine this possibility, 9 days after s.c. immunization, DLN RPMI 1640 culture medium as previously described (7). Then, 1 or 3 ϫ 107 ϩ cells from WT mice were incubated for 3 days with MOG35–55 CD4 T cells were purified with a negative selection kit on a MACS ϩ system (Miltenyi Biotec) and were transferred i.v. into naive and 500-rad peptide and then CD4 T cells were adoptively transferred i.v. into x-irradiated mice. X-irradiation was conducted using a MBR-1505R2 ir- WT and plt/plt mice. As shown in Fig. 1C, both WT and plt/plt radiator (Hitachi Koki). Where indicated, DLN cells from plt/plt mice were recipients developed EAE with 100% disease incidence with sim- stimulated with 10 ␮M MOG peptide in the presence of IL-23 (10 35–55 ilar clinical scores and time courses. As expected, DLN cells from ng/ml) and anti-IL-4 and anti-IFN-␥ mAbs (5 ␮g/ml each) for generating Th17 cells or in the presence of IL-12 (10 ng/ml) and anti-IL-4 and anti- immunized plt/plt mice did not develop EAE in naive WT mice IL-23 mAbs (5 ␮g/ml each) for generating Th1 cells. (Fig. 1C). These results strongly suggest that pathogenic T cells can infiltrate the CNS to induce EAE despite the absence of CCR7 Histological analysis ligands but that pathogenic cells fail to be generated in plt/plt mice On day 21 after immunization, animals were sequentially intracardially immunized with MOG35–55 peptide. perfused with saline containing heparin (Aventis) and 4% paraformalde- ␮ hyde. Spinal cords were embedded in paraffin. Transverse sections (5 m) Deficient IL-17 and IFN-␥ production by DLN cells from mice were prepared and stained with H&E. lacking CCR7 ligand expression In vitro recall response to MOG 35–55 To examine whether pathogenic cells were generated in plt/plt 5 DLN cells from immunized mice (5 ϫ 10 cells/0.2 ml/well) were cultured mice, we first compared the in vitro recall responses of DLN cells for 72 h with MOG . They were pulsed for 6 h with [3H]thymidine 35–55 from primed WT and plt/plt mice. DLN cells were prepared 9 days (1.25 ␮Ci/ml; Amersham Biosciences) and assayed for incorporation of [3H]thymidine using a Matrix96 direct beta counter (Packard Instrument). after immunization when EAE symptoms were not observed in Supernatants were collected at 24 h and assayed for IL-12 and IL-23 or at WT mice and 14 days after immunization when the symptoms 72h for IL-4, IL-6, IL-10, IL-17, TGF-␤, and IFN-␥ with OptEIA ELISA became evident. The proliferative recall responses to various doses kits (BD Biosciences). For intracellular staining, DLN cells were ␮ of MOG35–55 peptide were similar between DLN cells from WT incubated for 48 h with 10 M MOG35–55, followed by the addition of GolgiStop (BD Biosciences) for 5 h. Then the treated cells were stained and plt/plt mice prepared 9 days and 14 days after immunization with anti-CD4 and fixed and permeabilized using BD Biosciences Cytofix/ (Fig. 2, upper panels), suggesting T cell responses were similarly Cytoperm followed by intracellular staining for anti-IL-17, anti-IFN-␥,or elicited in WT and plt/plt mice. Next, we analyzed recall cytokine an appropriate isotype control. These cells were analyzed on a FACSCali- production to the MOG35–55 peptide. IL-4 and IL-10 were simi- bur flow cytometer (BD Biosciences). larly produced by DLN cells from WT and plt/plt mice (Fig. 2, Real-time RT-PCR second and third panels). Dose-dependent production of IFN-␥ or Total cellular RNA was isolated from cells using EASYPrep RNA (Takara IL-17 was detected in cultures of DLN cells from WT and plt/plt Bio). RNA (500 ng/reaction) was reverse transcribed using a High-Capac- mice, but production of each of these was severely di- ity cDNA Archive Kit (Applied Biosystems). For quantitative analysis, minished in plt/plt DLNs (Fig. 2, fourth and bottom panels). These The Journal of Immunology 2515 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. In vitro response to the MOG35–55 peptide of DLN cells from WT and plt/plt mice. WT and plt/plt mice were immunized as de- FIGURE 1. Failure of plt/plt mice and CCR7Ϫ/Ϫ mice to develop EAE. scribed in the legend for Fig. 1. DLN cells prepared 9 or 14 days after

A, Mice were s.c. immunized with MOG35–55 in CFA in the flanks and i.v. immunization were incubated with the MOG35–55 peptide at the indicated injected with PT on days 0 and 2 (10 mice/group). Clinical symptoms were doses and assessed for proliferation by [3H]thymidine incorporation on day monitored for 42 days after immunization. Mean clinical score Ϯ SD is 3. The amounts of IL-4, IL-10, IFN-␥, and IL-17 in the culture supernatants shown. Results from WT and plt/plt mice are shown in the left panel and were determined by ELISA using OptEIA kits (BD Biosciences). Each those from WT and CCR7Ϫ/Ϫ mice in the right panel. Representative re- result is expressed as the mean Ϯ SD. sults of three independent experiments are shown. B, The lumbar spinal cords were prepared from WT and plt/plt mice 21 days after immunization as described above and stained with H&E. Similar findings were obtained trast, as shown in Fig. 3, B and C, the expression of IL-23p19 from at least five different sections from each mouse. C, DLN cells were mRNA and IL-12p35 mRNA and production of IL-23 and IL-12 prepared from WT or plt/plt mice 9 days after immunization and incubated were much lower in the cells from plt/plt mice than in WT mice, ϩ ϩ with the MOG35–55 peptide for 3 days. WT CD4 T cells or plt/plt CD4 suggesting that the defect in production of IL-17 and IFN-␥ in ϫ 7 T cells (1 10 ) prepared from the treated cells were i.v. transferred into DLN cells from plt/plt mice was due to insufficient provision of naive and 500-rad x-irradiated WT or plt/plt mice (10 mice/group). Rep- IL-23 and IL-12. resentative results of three independent experiments are shown as the mean Ϯ EAE clinical score SD. Requirement for CCR-7 ligand in generation of IL-17 or IFN-␥-secreting T cells results suggest that plt/plt T cells could be primed by immuniza- Reduced in vitro IL-17 and IFN-␥ production by DLN cells from tion with the MOG35–55 peptide, but that the pattern of cytokine plt/plt mice suggested a defect in Th17 and Th1 cell generation. To responses differed from that of WT mice. examine this possibility, DLN cells were prepared 9 days after

For the optimal induction of IL-17-producing cells, IL-6, immunization, incubated with MOG35–55 peptide, and assessed for TGF-␤, and IL-23 are required (11–13). IL-12 is critical for in- intracellular IL-17 or IFN-␥ staining. As shown in Fig. 4A, ducing IFN-␥-producing cells (14). Deficient production of IL-17 CD4ϩIL-17ϩ Th17 cells were found at a much lower frequency in and IFN-␥ suggested that these cytokines were insufficiently pro- DLN cells from plt/plt mice than in those from WT mice (0.4% vs duced in DLN cells from plt/plt mice. As shown in Fig. 3A,in 4.2%). Addition of CCL19 or CCL21 to DLN cells from plt/plt

DLN cells prepared from WT and plt/plt mice 4 or 9 days after mice during incubation with the MOG35–55 peptide restored Th17 immunization similar levels of IL-6 and TGF-␤ production were cell generation from 0.4% to 3.0 or 4.1%, respectively (Fig. 4A). ϩ ␥ϩ observed following incubation with the MOG35–55 peptide. In con- Also, the frequency of CD4 IFN- Th1 cells was much lower in 2516 ROLE OF CCL19/21 IN IL-23 PRODUCTION AND EAE DEVELOPMENT

riched for CD4ϩ T cells, and transferred into WT mice. As shown in Fig. 5A, CD4ϩ T cells containing Th17 cells (CD4ϩIL- 17ϩcells, 9.2%; CD4ϩIFN-␥ϩ cells, 0.1%; Fig. 5C) induced EAE in the recipient mice with 100% disease incidence, whereas those containing Th1 cells (CD4ϩIL-17ϩcells, 0.1%; CD4ϩIFN-␥ϩ cells, 11.0%; Fig. 5C) did not, suggesting that Th1 cells are less efficient at inducing EAE. The cell preparation containing Th17 or Th1 cells was confirmed to predominantly produce IL-17 or IFN-␥, respectively (Fig. 5, D and E). The cells similarly prepared from WT mice and enriched for Th1 cells (CD4ϩIL-17ϩcells, 0.6%; CD4ϩ IFN-␥ϩ cells, 20.3%) also failed to elicit EAE in the recipient mice, whereas those containing Th17 cells (CD4ϩIL- 17ϩcells, 19.2%; CD4ϩIFN-␥ϩ cells, 0.4%) elicited EAE (Fig. 5B). These findings strongly support our interpretation that the defect in generating Th17 cells is crucial in the resistance to EAE development in plt/plt mice under the conditions used.

IL-23-dependent induction of pathogenic Th17 cells Downloaded from Deficient IL-23 production in DLN cells from plt/plt mice led us to evaluate the role of IL-23 in inducing Th17 cells. The addition of exogenous IL-23 to CD4ϩ DLN cells from immunized plt/plt mice stimulated with immobilized anti-CD3 and anti-CD28 mAbs in- creased the frequency of Th17 cells from 0.18 to 1.34% (Fig. 4B), supporting the idea that the defect in developing Th17 cells in plt/plt mice was due to reduced production of IL-23. To confirm http://www.jimmunol.org/ that stimulation with IL-23 was able to induce pathogenic T cells in EAE induction, DLN cells from immunized plt/plt mice were

incubated with the MOG35–55 peptide in the presence of IL-23, enriched for CD4ϩ T cells, and adoptively transferred into naive WT mice, which resulted in the development of EAE in the re- cipient mice (Fig. 5A). These results suggested that exogenous IL-23 was able to stimulate plt/plt mouse DLN cells along with the

MOG35–55 peptide to induce pathogenic Th17 cells, consistently, by guest on September 24, 2021 with the critical role of IL-23 in the induction phase of EAE (17). Taken all together, these findings suggest that the defect in plt/plt mice is likely a defect in Th17 cell generation due to deficient IL-23 production. FIGURE 3. Decreased production of IL-12 and IL-23 in DLN cells from plt/plt mice. DLN cells were prepared 4 or 9 days after immunization CCL19 and CCL21 stimulate DCs to produce IL-23 as described in the legend for Fig. 1. A, DLN cells were incubated for 3 DCs are known to produce IL-23 (18). The reduced production of days with MOG at the indicated doses. Culture supernatants were 35–55 IL-23 in the incubation of plt/plt DLN cells with MOG sug- assessed for TGF-␤ and IL-6. Results are plotted as the mean Ϯ SD. B, 35–55 ϩ gests the dependency of the IL-23 production on CCR7 ligands. To Expression of IL-23p19 mRNA and IL12p35 mRNA in CD11c cells was estimated by quantitative RT-PCR in DLN cells from WT and plt/plt mice. confirm this possibility, we prepared BMDCs and stimulated the The expression is shown as the mean Ϯ SD of the ratio to GAPDH, an cells with CCR7 ligands or other chemokines. LPS (100 ng/ml) internal control. These experiments were repeated five times with similar was used as a positive control for induction of IL-23p19 mRNA results. C, DLN cells from naive mice or 4 days after immunization were (18). CCL19 or CCL21 at 100 ng/ml increased IL-23p19 mRNA ␮ incubated with 10 M MOG35–55 for 24 h. Culture supernatants were as- expression, although not to the same extent as LPS (Fig. 6A, left sessed for IL-23 and IL-12. Results of triplicate assay are presented as the and middle). The chemokines CCL5 and CXCL12 did not stimu- mean Ϯ SD. late BMDCs to produce IL-23 (Fig. 6A, left). Confirming that CCL19 and CCL21 stimulate DCs through CCR7 to express IL- 23p19mRNA, BMDCs from CCR7Ϫ/Ϫ mice did not respond to the plt/plt mice than in WT mice (0.4% vs 4.4%). The addition of chemokines (Fig. 6A, right). CCL19 or CCL21 restored Th1 cell generation in plt/plt mouse We also assessed IL-23p19 mRNA expression by DCs in DLNs DLN cells from 0.4% to 3.1 or 3.2%, respectively (Fig. 4A). These in response to CCR7 ligands. DLN cells from immunized WT, Ϫ/Ϫ results support the hypothesis that the defect in generating Th17 or plt/plt, or CCR7 mice were incubated with the MOG35–55 pep- Th1 cells in plt/plt mice was due to the lack of CCR7 ligand tide for6hinthepresence or absence of CCL19 or CCL21. Then expression. CD11cϩ cells were enriched and assayed for IL-23p19 mRNA Previous reports demonstrated that the neuroantigen-specific expression. As shown in Fig. 6B (left), CD11cϩ cells from WT Th17 or Th1 cell is responsible for EAE induction (2, 15, 16). To mice expressed much higher IL-23p19 mRNA than those from determine which defect in generating Th17 or Th1 cells was more naive mice, and the addition of CCL19 did not further enhance critical in the resistance to EAE development, DLN cells from IL-23p19 mRNA expression in these cells from immunized WT

plt/plt mice were stimulated in vitro with the MOG35–55 peptide mice, probably because they had been exposed to CCL19 produced under the conditions for generating Th17 cells or Th1 cells, en- in DLNs. In CD11cϩ cells from plt/plt mice, however, the addition The Journal of Immunology 2517

FIGURE 4. Analysis of the T cell response in DLNs from WT and plt/plt mice immunized for EAE induc- tion and generation of Th17 or Th1 cells by the CCR7 ligand or IL-23. DLN cells were prepared from WT and plt/plt mice 9 days after immunization as described in the legend for Fig. 1. A, DLN cells were incubated with

MOG35–55 in the presence or absence of CCL21 or Downloaded from CCL19 (100 ng/ml), then assessed for intracellular IL-17 or IFN-␥ expression. Numbers in the right quad- rants are the percentage of the total cells. B, CD4ϩ DLN cells enriched from immunized plt/plt mouse were stimulated with immobilized anti-CD3 and anti- CD28 mAbs for 2 days in the presence or absence of

rIL-23 (10 ng/ml) and analyzed for intracellular IL-17. http://www.jimmunol.org/ C, DLN cells from plt/plt mice were incubated with the

MOG35–55 peptide for 2 days in the presence or absence of CCL21 or CCL19 alone or with anti-IL-23 mAb. The cells were analyzed for CD4 expression and intracellu- lar IL-17. by guest on September 24, 2021

of exogenous CCL19 or CCL21 increased IL-23p19 mRNA ex- T cells isolated from naive plt/plt mice or plt/plt mice primed with Ϫ/Ϫ pression (Fig. 6B, middle). As expected, cells from CCR7 mice the MOG35–55 peptide were not induced to produce IL-17 in re- did not respond to the addition of CCR7 ligands (Fig. 6B, right). sponse to immobilized anti-CD3 and anti-CD28 mAbs in the pres- CCR7 ligands also stimulated IL-23 production by BMDCs ence of exogenously added CCL19 or CCL21 (data not shown). from WT and plt/plt mice and by DLN cells from plt/plt mice (Fig. We concluded that CCR7 ligands stimulated DCs to produce IL- 6, C and D). DLN cells alone from immunized WT mice produced 23, which in turn resulted in Th17 differentiation. much more IL-23 than those from naive WT mice, probably be- cause endogenous CCR7 ligands induced a sufficient level of IL-23 Pathogenic T cell induction by incubation of DLN cells from production (Fig. 6D). Taken together, the results shown in Fig. 6 primed plt/plt mice with CCR7 ligands demonstrate that CCL19 or CCL21 is necessary and sufficient to To determine the pathogenicity of DLN T cells from plt/plt mice induce IL-23 production from DCs. Confirming that IL-23 pro- that had been incubated with CCR7 ligands under EAE-inducing duction in response to a CCR7 ligand plays a critical role in Th17 conditions, 9 days after immunization, DLN cells were incubated induction, in a dose-dependent fashion anti-IL-23 mAb inhibited for 3 days with the MOG35–55 peptide in the presence of CCL19 or Th17 cell generation following incubation of DLN cells from plt/ CCL21. CD4ϩ T cells were enriched from the treated cells and i.v. plt mice with the MOG35–55 peptide in the presence of CCL19 or injected into naive WT mice. As shown in Fig. 7A, the recipient CCL21 (Fig. 4C). mice developed EAE with Ͼ70% disease incidence. Thus, stimu- IL-12p35 mRNA expression and IL-12 production in BMDCs lation of DLN cells from primed plt/plt mice with the peptide in from plt/plt mice were also induced by the addition of exogenous the presence of a CCR7 ligand increased pathogenicity. CCL19 or CCL21 (data not shown). Finally, CD4ϩ T cells recovered from WT recipient mice were It was also possible CCR7 ligands directly stimulated CD4ϩ T analyzed for intracellular IFN-␥ and IL-17. DLN cells from primed ϩ ϩ cells to produce IL-17. However, this seemed unlikely since CD4 plt/plt mice (CD45.2 ) were incubated with MOG35–55 alone or in 2518 ROLE OF CCL19/21 IN IL-23 PRODUCTION AND EAE DEVELOPMENT

FIGURE 5. A Th17-enriched, rather than Th1- enriched, cell population was responsible for EAE development in recipient mice. A and B, DLN cells from primed plt/plt (A)orWT(B) mice were incu- bated with the MOG35–55 peptide for 3 days in the presence of CCL19, IL-12, and anti-IL-4 and anti- IL-23 mAbs for developing Th1 cells, in the pres- ence of CCL19, IL-23, and anti-IL-4 and anti-IFN-␥ mAbs for developing Th17 cells, or in the presence of IL-23 alone. CD4ϩ T cells (1 ϫ 107) prepared from the treated cells were i.v. transferred into naive and 500-rad x-irradiated WT mice (10 mice/group). EAE development is shown as a mean EAE clinical score Ϯ SD. C, Before transfer, Th1-skewed or Th17-skewed cells prepared from plt/plt mice as de- scribed above were assessed for intracellular IL-17 or IFN-␥ expression on a flow cytometer. Numbers in the right lower and left upper quadrants are the Downloaded from percentage of the total cells. D and E, Aliquots of these cells were incubated in the presence of

MOG35–55 and assessed for production of IL-17 (D) and IFN-␥ (E) as described in the legend for Fig. 2. http://www.jimmunol.org/

the presence of CCL21 or IL-23 and enriched for CD4ϩ T cells. In chemokines CCL19 and CCL21 play a critical role for the IL-23- these cells, IL-17ϩ cells were 0.1, 3.5, or 4.5% and IFN-␥ϩ cells were dependent generation of pathogenic Th17 cells, rather than in the 0.2, 4.7, or 0.4%, respectively (Fig. 7B, left column). Then, treated migration of pathogenic T cells into the CNS. cells were transferred into CD45.1ϩ WT mice. Fourteen days after, in IFN-␥ and IL-12 production was also decreased in DLN cells CD45.2ϩ spleen cells in the recipients of plt/plt DLN cells incubated from plt/plt mice (Figs. 2 and 3, B and C). The decrease in the

␥ϩ ϩ by guest on September 24, 2021 with MOG35–55 alone or in the presence of CCL21 or IL-23, frequency of intracellular IFN- CD4 T cells and in IL-12 pro- CD4ϩIL-17ϩ were 0.1%, 1.3%, or 1.2%, and CD4ϩIFN-␥ϩ T cells duction by DCs was recovered by the addition of exogenous were 0.1%, 1.3%, or 0.1%, respectively (Fig. 7B, right column). CCL19 or CCL21 (Fig. 4A and data not shown), as shown previ-

These results support that Th17 cells transferred survived in recipient ously (19), suggesting that the defect to generate MOG35–55-spe- mice at least until EAE was detectable in the recipient mice and could cific Th1 cells could be included in the resistance to EAE devel- be pathogenic for EAE development. opment in plt/plt mice as reported very recently (15, 16). However, this possibility is unlikely since DLN cells prepared from plt/plt Discussion mice and incubated under the conditions for generating Th1 cells In the present study, mice lacking the expression of the CCR7 could not develop EAE in naive WT recipient mice, whereas those ligands CCL19 and CCL21 ( plt/plt mouse) or lacking expression incubated under the Th17-generating conditions did develop EAE of CCR7 failed to develop EAE following a standard immuniza- (Fig. 3B). Based on these findings, we conclude that the failure to tion protocol with which WT mice developed EAE. The depen- develop EAE in plt/plt mice was due to a defect in generating dency of EAE development on CCR7 ligands is not due to a defect IL-23-dependent Th17 cells. The reason for the discrepancy be- in the migration of pathogenic T cells in plt/plt mice, since adop- tween our results and those in which IL-12-dependent Th1 cells tive transfer of pathogenic CD4ϩ T cells prepared from DLN cells are able to transfer EAE (16) is presently unknown. It might be due of WT mice result in EAE development in plt/plt and WT recipient to different conditions for immunization. Our priming mice once, mice with similar time course and disease severity. Instead, a de- not twice, with the MOG35–55 peptide might be suboptimal for fect was observed in the ability of mice lacking CCL19 and EAE induction, which resulted in the inability for Th1-skewed T CCL21 or lacking CCR7 to develop pathogenic Th17 cells. Con- cells to transfer EAE to recipient mice, as described previously sistently, expression of IL-23p19mRNA and IL-23 production (15). Th17 cells have been shown to induce greater encephalito- were decreased in DLN cells from immunized plt/plt mice (Fig. 3). genic responses on a cell-cell basis, following passive transfer, IL-23 has been shown to be a critical Th17 growth and survival when compared with Th1 cells (2). Several recent reports support factor (11–13). The expression of IL-23p19mRNA and production the interpretation that pathogenic Th17 cells induce EAE more of IL-23 by DCs from plt/plt mice were recovered by the addition efficiently than IL-12-dependent Th1 cells (15, 17, 20–22). of exogenous CCL19 or CCL21 (Fig. 6). T cell responses to the In a previous report where mice were immunized twice with the

MOG35–55 peptide in DLNs from plt/plt mice were not too low to MOG35–55 peptide in CFA and injected i.v. with PT on days 7 and generate pathogenic T cells. Proliferation and IL-4 production in 9, CCR7Ϫ/Ϫ mice developed EAE with very similar disease se- recall responses were quite similar in plt/plt and WT mice. We verity to WT mice (23). In plt/plt mice, we observed EAE devel- conclude that the failure to generate pathogenic T cells in plt/plt opment with two immunizations of the MOG35–55 peptide, but the mice was due to a defect in induction of pathogenic effecter T cells mean clinical score for disease severity in plt/plt mice was less rather than an insufficient response to the MOG35–55 peptide. Thus, than half that of WT mice (data not shown). The reason for the The Journal of Immunology 2519 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 6. CCR7 ligands stimulate DCs to express IL-23p19 mRNA and to produce IL-23. A, BMDCs were prepared from WT, plt/plt, and FIGURE 7. Restoration of pathogenic T cells by incubation with CCR7 ϩ CCR7Ϫ/Ϫ mice and stimulated with LPS or the indicated chemokines at ligands. DLN cells from immunized plt/plt mice (CD45.2 ) were incu- ϫ 6 ␮ 100 ng/ml for 6 h. Cellular RNA was prepared from each cell population bated at 4 10 cells/ml with 10 M MOG35–55 peptide in the presence ϩ 7 and IL-23p19 mRNA expression was evaluated by quantitative RT-PCR. of CCR7 ligands (100 ng/ml) for 3 days. A, CD4 T cells (3 ϫ 10 ) The expression is shown as the mean Ϯ SD of the ratio to GAPDH, an prepared from the treated cells were i.v. transferred into naive and 500-rad internal control. B, DLN cells were prepared 4 days after immunization x-irradiated WT mice and mice were monitored for EAE (10 mice/group). from WT, plt/plt, and CCR7Ϫ/Ϫ mice and incubated with 10 ␮M A mean Ϯ SD of EAE clinical score is plotted. The EAE incidence was 0% for recipients of cells incubated in the absence of CCR7 ligands, 70% for MOG35–55 peptide in the presence or absence of CCL19 or CCL21 for 6 h. CD11cϩ cells were enriched with a positive selection kit (BD Biosciences) those in the presence of CCL19, and 80% for those in the presence of by MACS. CD11cϩ cells were 89.2, 92.2, and 90.4% for WT, plt/plt, and CCL21. Data representative of three independent experiments are shown. ϩ CCR7Ϫ/Ϫ mice, respectively. Cellular RNA was prepared from each cell B, CD4 T cells prepared from the treated cells were analyzed for expres- population and assessed for IL-23p19 mRNA expression by quantitative sion of intracellular IL-17 and IFN-␥ on a flow cytometer (pretransfer). ϩ RT-PCR. Controls were LN cells from naive mice. Expression is shown as They were transferred into CD45.1 C57BL/6-Ly5.1 mice. Fourteen days the mean Ϯ SD of the ratio to GAPDH as an internal control. C and D, after (posttransfer), spleen cells from recipient mice were prepared and ϩ BMDCs (C) or DLN cells (D) from WT and plt/plt mice were stimulated donor cells (CD45.2 ) were gated and analyzed as above. Numbers in the ϩ as described above for 24 h. The supernatants were assessed for IL-23 left upper and right lower quadrants are the percentage of the total CD4 ϩ using an ELISA kit. Results are shown as the mean Ϯ SD of triplicate or CD45.2 cells for pretransfer or posttransfer, respectively. assays.

stimulation with CXCL9, CXCL10, and/or CXCL11 via CXCR3, discrepancy between our results and those by Pahuja et al. (23) is an additional receptor for mouse CCL21 (25–27), might partially presently unknown; however, low-level expression of CCL21- substitute for CCR7 stimulation in heavily immunized plt/plt or leucine in plt/plt mice (24) might account for the discrepancy. The CCR7Ϫ/Ϫ mice. These possibilities might also explain why DLN difference in immunizing protocols also might be an explanation. cells from plt/plt mice produced a small but detectable amount of Repeated immunization of plt/plt mice may increase production of IL-17 (Fig. 2). IL-6 and TGF-␤, thereby contributing to further generation of Failure to develop EAE in plt/plt mice was due to the inefficient Th17 cells and development of EAE (9, 11, 12). Alternatively, generation of Th17 cells, which mainly resulted from deficient 2520 ROLE OF CCL19/21 IN IL-23 PRODUCTION AND EAE DEVELOPMENT

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